Adaptive Evolution in Aminoacyl-tRNA Synthetases Drives Antibiotic Tolerance and Resistance in Clinical Klebsiella pneumoniae Isolates.

Klebsiella pneumoniae is a major global health threat due to the rapid spread of antimicrobial resistance (AMR), which severely limits treatment options. Although horizontal gene transfer of mobile genetic elements is a key driver of multidrug resistance, this study explores how adaptive evolution within the core genome may also contribute to the clinical success of resistant strains. Using whole-genome sequencing-based pangenome analysis of clinically relevant classical K. pneumoniae isolates, we identified 3159 variants distributed across 414 core genes, indicating that even highly conserved cellular functions accumulate mutations in clinical environments. Functional enrichment analysis revealed a significant concentration of mutations in the aminoacyl-tRNA synthetase (aaRS) pathway, a central component of protein synthesis and a known antibiotic target. Multiple missense variants were detected in eight aaRS genes, with the Asn366Asp mutation in metG present in all analyzed isolates. We hypothesize that this conserved, recurrent mutation may contribute to bacterial adaptation, potentially by modulating protein synthesis, although this proposed mechanism remains speculative and requires experimental validation. Recurrent mutations were also observed in ileS and leuS, both targets of existing antimicrobials, while a Ser480Pro mutation in pyrG (CTP synthase) was identified in several isolates. Overall, these findings highlight core genome variation as a potential contributor to antimicrobial resistance in K. pneumoniae and suggest that conserved mutations such as metG Asn366Asp may represent candidate genomic biomarkers warranting further investigation. Because this study is based solely on comparative genomics, the proposed functional and mechanistic interpretations should be regarded as hypotheses for future experimental testing.